} Dear Microscopists, } I am helping a student set up a cryofixation (plunge or slam on a KF80) and } cryosubstitution project on blades of rye grass. She hopes to immunolabel } for CI proteins in the blade....There doesn't seem to be much botanical } cryofixation/crysubstitution literature out there. } Can anyone offer some suggestions? } ... As a starting point, try:
Zhang G.F. and L.A. Staehelin. 1992. Functional compartmentation of the golgi-apparatus of plant-cells - immunocytochemical analysis of high-pressure frozen-substituted and freeze-substituted sycamore maple suspension-culture cells. Plant Physiology 99:1070-1083.
Hippe-Sanwald, S. 1993. Impact of freeze substitution on biological electron-microscopy. Microscopy Research and Technique 24:400-422.
Galway M.E. 1993. Ultrastructure of the endocytotic pathway in glutaraldehyde-fixed and high-pressure frozen freeze-substituted protoplasts of white spruce(picea-glauca) Journal of Cell Science 106:847-858.
} Dear Microscopists, } I am helping a student set up a cryofixation (plunge or slam on a KF80) } and cryosubstitution project on blades of rye grass. She hopes to } immunolabel for CI proteins in the blade. This protein is a Poty } virus-induced protein. Our problem is that we can't find much published } material suggesting the best substitution medium, best fixative and } recommended Lowicryl to use. There doesn't seem to be much botanical } cryofixation/crysubstitution literature out there. } Can anyone offer some suggestions? } } Allan Mitchell } Technical Officer } South Campus EM Unit } Dept of Anantomy and Structural Biology } Otago Medical School } NZ } } Richard Easingwood } Department of Anatomy and Structural Biology, } P.O. Box 913 } University of Otago, } Dunedin, New Zealand. } Fax:64-3-479 7254 } Telephone:64-3-479 7301 } ====== Here are a few references:
M.K. Kandasamy, M.V. Parthasarathy and M.E. Nasrallah, 1991. High Pressure Freezing and Freeze-substitution improve immunolabeling of S-locus specific glycoproteins in the stigma papillae of Brassica. Protoplasma 162: 187-191
Biao Ding, J.S. Haudenshield, R.J. Hull, S. Wolf, R.N. Beachy anf W.J. Lucas, 1992. Secondary Plasmodesmata are Specific Sites of Localization of the tobacco mosaic virus movement protein in transgenic tobacco plants. The Plant Cell 4: 915-928.
Some references for Lowicryl embedding of cryofixed plant material:
Document 1 TI IMMUNOCYTOCHEMICAL LOCALIZATION OF NUCLEAR ANTIGENS IN THE UNICELLULAR GREEN ALGA CHLAMYDOMONAS-REINHARDTII PROCESSED BY CRYOFIXATION AND FREEZE-SUBSTITUTION AU TREMBLAY-S-D. LAFONTAINE-J-G. SO PROTOPLASMA 165 (1-3). 1991. 189-202.
Document 2 TI FLAVONAL RING B-SPECIFIC O GLUCOSYLTRANSFERASES PURIFICATION PRODUCTION OF POLYCLONAL ANTIBODIES AND IMMUNOLOCALIZATION AU LATCHINIAN-SADEK-L. IBRAHIM-R-K. SO ARCH BIOCHEM BIOPHYS 289 (2). 1991. 230-236. Document 3 TI IMMUNOCYTOCHEMICAL DEMONSTRATION OF THE PEROXISOMAL ATPASE OF YEASTS AU DUMAS-E. LHERMINIER-J. GIANINAZZI-S. WHITE-R-F. ANTONIW-J-F. SO J GEN VIROL 69 (10). 1988. 2687-2694. (This article also has localization in plant cells) M.V. Parthasarathy Section of Plant Biology, 228 Plant Science Building Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734; Fax: 607-255-5407 E-Mail: mvp2-at-cornell.edu
Two who have done good work on cryosubstitution with plants are Rick Howard at DuPont (howardrj-at-esvax.dnet.dupont.com) or Howard Berg at Memphis State U. (Bergrh-at-msuvx1.memphis.edu) ********************************************************** * Greg Erdos ** * * Director, ICBR EMCL ** Phone 904-392-1295 * * 218 Carr Hall ** FAX 904-392-8598 * * University of Florida ** gwerdos-at-gnv.ifas.ufl.edu * * Gainesville, FL 32611 ** * **********************************************************
You asked about azocarmine. I have not found a specific reference to azocarmine, however, the azo dyes are very common. Examples of azo dyes are congo red, CI Fast Red, CI resorcine dark brown, and CI naphthol blue black B. The general reaction is:
ArN2+X "+" HR to ArN=NR "+" HX
I do not know for sure, but carmine is an old art color for red. I would bet you have an old jar of something similar to Congo Red. If you want to read more about the way azo dyes work, there is a very good section in Kirk Othmer's Encyclopeadia of Chemical Technology, 3rd ed. Vol 3, Vol 3, "Azo Dyes".
Melanie Thom, Sr. Chemist AlliedSignal Aerospace South Bend, IN 219-231-2856
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G'day Fellow Microscopy Subscribers:
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name will be registered I will send you all an update of this as soon as possible.
The program which best meets your criteria for area and perimeter measurement and cost is NIH Image. You will be pleasantly surprised at its capability and reliability. According to the manual, it is available from:
1) From a friend. The Image program, including source code and documentation, is public domain and may be freely copied, distributed and modified. However, if you modify Image, please update the about box before distributing your version of the program.
2) Via anonymous FTP from zippy.nimh.nih.gov[128.231.98.32]. Enter "anonymous" as the user name and your e-mail address as the password. The /pub/nih-image directory contains the latest version of Image (nih- image154_fpu.hqx or nih-image154_nonfpu.hqx), documentation in Word format (nih- image154_docs.hqx), and complete Think Pascal source code (nih- image154_source.hqx). The directory /pub/image/images contains sample TIFF and PICT images. The directory /pub/image/image_spinoffs contains versions of Image extended to do FFTs (ImageFFT), fractal analysis (ImageFractal), and to support quantitative evaluation of cerebral blood flow, glucose metabolism, and protein synthesis (Image/MG). There is a README file (0README.txt) with information on the file formats used.
3) Library 9 (Graphics Tools) of the MACAPP forum on CompuServe. Source code is in Library 6 of the MACDEV forum.
4) Twilight Clone BBS in Silver Spring, MD. The Clone has 16 lines on sequential rollover, starting with 301-946-8677. To guarantee a V.32 connection, call 946-5034. Image is currently available at no charge from the Twilight Clone.
5) Subscribe to the NIH Image mailing list by sending a message containing the line "subscribe nih-image {your name} " to listserv-at-soils.umn.edu. Next obtain a list of the available NIH Image archive files by sending an "index nih- image" command to listserv-at-soils.umn.edu. These files can then be retrieved by means of a "get nih-image filename" command. The files are Binhexed and broken into chunks less than 32K in size. The NIH mailing is maintained by the Soil Science Department at the University of Minnesota.
6) NTIS (National Technical Information Service), 5285 Port Royal Road, Springfield, VA 22161, phone 703-487-4650, order number PB93-504868 ($100 check, VISA, or Mastercard). Both the zippy.nimh.nih.gov FTP site and the Twilight Clone BBS are likely to have newer versions of Image than NTIS.
Azocarmine, despite what the name suggests is not an azo dye but a member of the quinone-imine group (azin subgroup). Azocarmine G is a sodium salt of a disulfonic acid of phenylrosinduline and has a C.I. number of 50085. There is a related dye, azocarmine B which is a trisulfonate (C.I. 50090).
I have been able to find little on the use of the dye as a stain apart from it being used as an alternative to acid fuchsin in Mallory's connective tissue stain and a couple of general purpose stains.
Most of my information comes from a volume entitled "Biological Stains" by H.J. Conn 7th edition 1961 which is a good guide to a wide range of dyes used for biological staining and was prepared in association with the Biological Stain Commission. I assume there are later editions out there somewhere but this is the latest in our library.
Hope this is of some help
Ian Hallett HortResearch Mt Albert Research Centre Auckland New Zealand
Dear microscopists, In June I posted a note asking if anyone knew of an alternative source of targets for Biorad sputters coaters.I had had problems getting targets within a reasonable period of time through Fisons (the last one took six weeks to come). This is not a problem unique to us judging by the response I got on this listserver. David Henriks suggested Testbourne Ltd at Unit 12, Hassocks Wood, Stroudley Road, Basingstoke, Hampshire RG24 0NE, England. I followed this up and they are now able to supply these annular targets (Au or Au/Pd) at price very competitive to Fisons. They have also given us a discount for the little remaining target material on our used targets which we sent them, if you are like us you will have lots of old targets so its good to find a home for them.
Needless to say I have not the slightest commercial interest in Testbourne Ltd.
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
RE: Cryofixation/cryosubstitution of plant material
Many thanks to all who responded to my request. The response has been fantastic. I will report on our efforts as soon as we have got our technique to work.
Allan Mitchell
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
We have recently started to become more aware of the purity of our glutaraldehyde however a brief excursion into this form of quality control has left me confused. We have analysed four different supplies of 25% glutaraldehyde with the following results: (all stock solutions were kept in the refrigerator at 4deg C)
Stock A: received into the Unit Jult 1993 (500ml bottle of EM grade). Spectrophotometric result:high 235nm absorbance peak with resulting P/M ratio of 3.18 pH 5.16 osmol of 1% solution 119 mosmol/kg
Stock B: received into the Unit May 1994 (500ml bottle of EM grade). Spectrophotometric result:high 235nm absorbance peak with resulting P/M ratio of 3.08 pH 5.24 osmol of 1% solution 127 mosmol/kg
Note: stocks A and B are from the same supplier.
Stock C: received inot the Unit June 1994 (ampule of 25% under nitrogen, 10mls). Spectrophotometric result:low 235nm absorbance peak with resulting P/M ratio of 0.22 pH 3.31 osmol of 1% solution 103 mosmol/kg
Stock D: received into the Unit June 1994 (100 ml bottle) Spectrophotometric result:low 235nm absorbance peak with resulting P/M ratio of 0.18 pH 3.66 osmol of 1% solution 108 mosmol/kg
The P/M ratio is the ratio of polymer ('impurities', absorbance peak at 235nm) to monomer (good stuff, absorbance peak at 280nm).
I am confused because in the literature some say that glutaraldehyde with a baseline P/M ration of 0.2 or more should be rejected (so our stocks A and B should be thrown out). However others report that if the pH drops below 3.5 then it should be discarded.
In our test the two stock solutions with the best P/M ratios have the worst pH readings and vice versa. Do I throw the whole lot out and go fishing?
Brenda Weakley (J of Microscopy, Vol 101, July 1974 p127) reports;1)Low pH of the stock solution is the most detrimental factor in fixation and 2) a high 235nm/280nm ratio gives poor fixation.
I am going to run an experimental trial with these fixatives on tissue however I am interested to hear others' thoughts and opinions on this topic.
Allan Mitchell
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
A friend in one of the clinical sciences has a tissue section he wants to determine the Pb levels in. Does any one know of a commercial operation that could give him some help?
Thanks.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
I am trying to get my own copy of Electron Microscopy of Thin Crystals by Hirsch et al.(1977 edition) but the book shops I have tried so far have not been very optimistic. Does anyone know of a (specifically) scientific second hand/out of print book shop in the UK, USA or even here in Canada which could help me?
Thanks Ciara Mullan CEMD McMaster University 1280 Main St West Hamilton Ontario L8S 4L7
We are in process of getting a new autoproessor for our EM film.
Currently we have a Labtronix Autoprocessor 1400 which we had purchased from Energy Beam Sciences Inc. 7-8 years ago, and are unhappy with it due to numerous problems and down time.
Are there any out there that are totally automatic in cycle and are made out of stainless steel or some sort of metal?
Any suggestions welcomed.
Raj
Raj-at-Bioimg.umdnj.edu Robert Wood Johnson Medical School Dept. of Pathology (908) 235-4648
Hi, I have been fixing microspheres (100um diameter average) made up of polysaccharides for SEM and I have encountered deformation particularly in the critical point apparatus. To overcome this I put the spheres in a small plastic cylinder that has a removable mesh cover at both ends, so the liquids can flow in and out of it without problems. This small device fits nicely in our polaron critical point dryer and you can purchase them at: SPI supplies PO BOX 656 569 EAST GAY STREET, WEST CHESTER, PA, 19381-0656, USA FAX 1 215 436 5755 PHONE 1 215 436 5400 PRODUCT: MICROPOROUS SPECIMEN CAPSULES # 13215
PELCO INTERNATIONAL PO BOX 492477, REDDING, CA, 96049-2477, USA PHONE 1 916 243 2200, FAX 1 916 243 3761 PRODUCT:CAPILLARY TUBE TISSUE PROCESSING #102, # 6GN200 MICRO TISSUE CAPSULE ASSEMBLY #1025...
Ciara Mullan at Mc Master University was looking for a copy of ELECTRON MICROSCOPY OF THIN CRYSTALS BY HIRSCH ET AL. I have found a number of good books at this location: (I am not affiliated in any way) This place has many books on Electron Microscopes, antique scientific instruments, primarily microscopy. The address is: RThe GemmaryS P.O. Box 816 Redondo Beach CA. 90277 310-372-5969 Hope they have it. Larry Albright
I am restarting the Listserver this morning. Hopefully most of the problems have been cured. The listserver is now operating on a new computer It's name has not yet been registered on the International Domain Nameserver System,. For the present time mail sent to ANLEMC.MSD.ANL.GOV will be forwarded to this system and then sent out. I expect that by tomorrow at the latest it's host name
The computer workshop & software exchange went well again this year. We had 10 computer (6 Macs and 4 PC's networked together and available for use from ~ Noon Monday through ~ Noon Friday. 516 people registered at the door and had access to over 500 Mbytes of software which was distributed via the EMMPDL, MASLib and Freeware/Shareware libraries maintained by John Mansfield (U. of Mich) and myself.
Registrants, in addition to bringing their own disks, purchased the entire 500 disk supply brought by students of the American Nuclear Society who were selling them at the workshop for the benefit of participants.
5 New things that occured this year:
1.) Announcement of Anonymous FTP sites for accessing the Software Libraries.
www.amc.anl.gov (ANL) freebie.engin.umich.edu (U of Mich)
2.) Announcment of a World Wide Web Sites for Microscopy & Microanalysis with Society Information.
http://www.amc.anl.gov (ANL) http://www.engin.umich.edu/~jfmjfm/mas_folder/mashomepage.html (U of Mich)
3.) On line computer tutorials on Image processing and telecommunications. These were video taped and will be avaiable through the MSA video tape library at the MSA Business office.
4.) On line Internet connections via a PPP phone link through Tulane University.
5.) Round Robin Grayscale Printer Test.
As proposed by J. Chandler & S. McKernan, we ran a round robin grayscale printer test this year. Two test images were placed on an FTP site (AAEM.AMC.ANL.GOV) and were downloaded by 14 individuals and printed on 15 different printers (with some duplicates).
Thanks are due to the following individuals & 3 commerical firms (in semi-random order) for spending their time and effort to make this round robin test a success.
G. Nord - USGS C. Wood - Dow S. McKernan - Univ. of Minn. J. Neilly - Abbott Labs. M. Bisher - NEC M. Disko - Exxon P. Melvile - ? R. Hermann - ETH Zurich J. Chandler - Colorado State Univ. J. Mansfield - Univ. of Mich N. Zaluzec - ANL Kodak Condonics Polaroid
The following printers were represented:
Laser & InkJet
Apple Laserwriter NTX Apple Laserwriter Pro 630 QMS 815 MR HP LaserJet 4ML HP DeskJet 1200C/PS LaserMaster Unity 1200 XL
These printers ranged from conventional Laser printers & Ink Jets to full blead Dye Sublimination printers or equivalent.
Some pronounced differences were apparent especially in cases where 2 prints from the same printer were contributed from different places but with strikingly different results. The conclusion was that the differences were attributed to the printer drivers used in the computer, rather than to the printers themselves.
We did not conduct a survey of the opinions of the people who inspected the results, however, it was clear (to me) that as expected not all the printers were equivalent. Most of the Laser printers showed a clear dithering pattern. The dye sublimination printers generally showed good tonal response, but some had definite tendancies to show a slight off-color response. For example, some had a slight bluish to green tinge, others did not have good "blacks". Not all the printers showed the ability to reproduce some of the high resolution test images and text output also varied as small fonts were not always reproduced on some models.
In order to continue the test, I have taken all the prints home with me and intent to store them in a file cabinet for the next year. I will bring each of them back to next years workshop (Kansas City) and ask each of the original contributors to send a new fresh copies of the same image (the master image will be kept on file here at the FTP site at ANL). This way we will be able to test for long term stability of the prints.
Next year, if there is still interest we will may take an opinion survey to see what the participants of the workshop think of the output. This years test was just to see what might be done.
By the way, if anyone else still wants to contribute an output from their printer. Download the test images from AAEM.AMC.ANL.GOV and put them in the mail to me. I will store them with the rest for next year.....
If there is still interest we may extend the test to include color.
- Overall it was a busy meeting and reasonably successful workshop -
Cheers - Nestor J. Zaluzec ANL
P.S. I enjoyed touching base with a lot of the Microscopy Listserver subscribers who introduced themselves at the meeting it's always good to associate a real person with the Email messages which come down the line.....
We have got a problem for ES-423E LaB6 filament (style 90-15) bought from The Kimball Physics.
We have a Jeol-2000FX TEM. As our TEM samples are mostly metals, the TEM always works at 200 KV.
We used to use Denka LaB6 Cathode and the emmission of the cathode was 30 micro-Amp.. The life span of the Denka filament was about 500 - 1000 hrs.
We recently changed our cathode with ES-423E LaB6 filament (Style 90-15, The Kimball Physics) which is supposed to last longer than Denka one. The emmission of the filament was kept at 16 micro-Amp.. However, after about 240 hrs, the filament has so seriously run up that we have to change the filament to keep the microscope running. We checked the vaccum system and found nothing wrong. The bias mode and beam current also looks OK.
Is there anyone used the ES-423E filament? We do appreciate any suggestions and comments on this issue.
Thanks
X. Li (Uni. of Wollongong, Australia) P. Renwick (BHP, Australia)
The lifetime of an LaB6 filament depends a lot upon how it is first heared up, as well as design considerations such as the sharpness of the tip. In general, if you heat them up VERY SLOWLY, i.e. over about 15mins to an hour, you should (we do) get lifetimes } 2000 hrs. At this level the sharpness of the tip and its slow blunting due to attack by oxygen etc (which you cannot avoid except with UHV) and sublimation becomes important.
The key issue, SEM or TEM as far as I know is to desorb oxygen (or oxygen containing gases) at low temperatures so as to minimize/eliminate reaction with the LaB6 filament at higher temperatures which produces an oxide layer.
Following along on Prof. Marks comments on saturating a LaB6 in a TEM, does the 15-60 minutes include pre-conditioning the filament. On the surface, these long times for powering up appear to be a waste of money. I don't know what filaments cost, but I would wager a bet that it would not take too many hours of powering up the filament very slowly to have the hourly charge of the TEM exceed by several times any savings seen by stretching the lifetime of the filament by a factor of 3 to 5. Have you done a cost analysis, including down time and pain-in-the-butt factors during filament swaps?
Message-Id: {9408081606.AA04250-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Sample Mounting for Tripod Polishing Orig-Sender: {CINIBUMK-at-ml.wpafb.af.mil}:ddn:wpafb Orig-Author: {CINIBUMK-at-ml.wpafb.af.mil}:ddn:wpafb ----------------------------------------------------------- I've been trying to prepare e-transparent samples of ceramic fibers embedded with epoxy by tripod polishing on diamond impregnated films, a la IBM. As they suggest we have been mounting the samples with super glue to a glass slide, which is then mounted with wax to the SS stub. The problem is that the super glue is debonding from the glass slide. I've tried slightly roughening the glass slide with 600 grit SiC paper and also carefully cleaning the glass prior to bonding. Thisd does not seem to help. The glue always remains bonded to the sample. Does anyone have suggestions for improving the bonding with superglue or any other glue or wax that will hold up and not flake off during the final polishing of thin sections. HAs anyone tried crystal bond? I am new to this technique and welcome the advice of those with more experience in the art.
Mike, if your problem is superglue delamination off of the glass slide, then you are probably just not roughing the slide surface up enough. Don't worry about the slide and grind it until you have a nice translucent surface; the refraction translucence will basically disappear once you have put the superglue on. I went through the same difficulty a few years ago. You can also freely use a larger grit, say 180 or 240 for grinding. Make sure that you grind in multiple directions as well, as I suspect that a large part of the secret is essentially mechanical interlocking.
Daniel L. Callahan Department of Mechanical Engg. and Materials Science Rice University dlc-at-owlnet.rice.edu
Netters, Richard Easingwood asked a series of interesting questions about purity and qulaity of glutaraldehyde. One of the issues was the pH of the glut. My understanding is that glut has relatively little buffering capacity and so that when you add this aldehyde to your buffered solution, the pH of the solution won't be affected. Is this wrong? Does anybody actually know what the buffering strength of glut is? Thanks, Tobias Baskin
Dear Xin Yang; If you have access to a large chamber SEM with good 5 kv. or lower operating capability, you may find it interesting to examine the LaB6 filament in the SEM. There are several factors that could account for the low life time (240 hrs.) of this filament. Among these are: 1.) Defective when installed, 2.) heated to rapidly, 3.) poor vacuum in gun chamber, 4.) lack of pre heating, and even the possibilty that 5.) a sample exchange was made with the filament saturated.
Although a new filament can be defective, this is very unlikely so one of the other possibilities is the cause. If you can examine the filament in the SEM, you will be able to measure the specified parameters of the filament and determine if the material loss is excessive. A new LaB6 KPI has a diameter of about 320 microns. Check with KPI to verify this and then calculate the loss. Acceptable rates of loss are about 0.1 micron per hour in the worst of cases. You only need a magnification of about 250x lookng straight down at the tip for this measurement. You should also increase the magnification to about 2000x to examine the the tip to see if it still has the correct characteristics.
Especially look for chunks of material missing from the crystal. If there are such defects, this would indicate poor vacuum or a slight vacuum loss while the filament was heated. Do you have a vacuum monitor in the gun chamber? The pressure in the gun should be in the low 10-7 torr range.
If you have access to such an SEM, A convenient holder for the filament can be made from the original base that is used for shipping, by simply cutting off the threaded end. This will fit into a conventional SEM mount like the 32 mm. holder for a JEOL 35 or 820/840 series SEM. Just make sure that you have sufficient Z in the SEM and don't use an chamber interlock if you have one
Hope this helps. If you have an opportunity to try this I would be interested in what you find.
Dear microscopists, Thanks very much to all who responded a week or two ago re my request for peoples' feelings about their print processors. Having read your replies and looked at the Agfa Pro, Agfa Rapiline and the Durst modular unit 'in the flesh' we are tending towards the Rapiline.Unfortunately no one who replied mentioned this model. Several people had reservations about the Ilford 2150 processor (although admittedly some loved it). If anyone has any particularly bad (or good) experiances with the Rapiline I would like to hear from you. Yours faithfully,
Richard Easingwood Department of Anatomy and Structural Biology, P.O. Box 913 University of Otago, Dunedin, New Zealand. Fax:64-3-479 7254 Telephone:64-3-479 7301
With reference to the questions asked by Richard Easingwood and Tobias Baskin: Some of the questions regarding the pH of Glut and its buffering capacity were addressed in a paper by Coetzee & Van der Merwe - Some characteristics of the buffer vehicle in glutaraldehyde-based fixatives (Journal of Microscopy, 146, 143-155, 1987). The conclusion reached in this paper was that you need - very approximately - at least 50mM of buffer to adequately stabilize the pH of a 2.5% Glut solution. The problem is in defining what is adequate for your needs. As little as 2% of Glut added to a 0.2M buffer causes a discernable drop in pH. This is true for a wide variety of buffers around pH 6.5 - 8.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
Can anyone tell me where I might find a history of EM? I know that Ernst Ruska developed the Electron Lens and recieved the Nobel prize in 1986. I would like some more information on things like: when glass and diamond knifesw were first used. When Epon was developed. When were certain structures first observed like; synapes, microfilaments, or or other things that could not be seen with LM. I am sure that all this information is preserved in different publications but our medline does not allow for searchs back that far. If there is not a "History of EM" some one should write one. Any help on this would be greatly appreciated.
Lary Hawkey That is Larry Hawkey (not Lary) hawkey-at-neuro.duke.edu
Anyone, I am looking for papers which deal with the degradative effects of E beams on cellulose structures. In particular - we are examining single wood fibers under tension inside an ESEM. We are using quite low accel. voltages c. 15KeV. Statistically it looks like the strength of the fibers is unaffected - the tests are over within a minute or so. However, something must be happening somewhere in the fiber wall (hemicellulose maybe) because after exposure to the beam for a little longer we are experiencing some burning. Any body got any words of wisdom (mine is limited) on this. Any comments or better still references, would be appreciated.
Hello, I was wondering if anyone had any experience with x-TEM of 6H-SiC,3C-SiC, on each other and Si. We are trying to look at the interface between SiC-Si and 6H-3C. Any thoughts on preparation (right now we cryo-ion mill) due to the hardness would be appreciated.
Sterling P. Newberry put together a nice history of EM, "EMSA and Its People, The First Fifty Years". 1992 by the Electron Microscopy Society of America, LC#92-72571.
Thanks for all the responses for suppliers of abrasive diamond films. I have compiled a list of the suppliers recommended to me and have included the list with this message. I meet with most of these suppliers while at the MAS meeting in New Orleans, and along with the diamond films they have other supplies that will come in handy for thin foil specimen preparation. Give 'em a call if you don't already have one of their catalogs.
thanks again for all the recommendations, John
replies for suppliers of diamond films:
1. Allied High Tech Products P.O.Box 4608 2376 East Pacifica Place Rancho Dominguez California, 90220 Contact: Clayton A. Smith Phone: 1-800-950-9347
3. Buehler Ltd. 41 Waukegan Rd P.O. Box One Lake Bluff, IL 60044 (708)295-6500 X4546.
4. South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673 TEL: 800-728-2233 714-492-2600 FAX: 714-492-1499
5. Imperial Lapping Films (reference below) 3M Corp. 3M Center St Paul, MN."
The title of the paper is "Recent Developments in The Use of The Tripod Polisher for TEM Specimen Preparation", by John Benedict, Ron Anderson and Stanley J. Klepeis.
Message-Id: {9408101624.AA23224-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: x-SiC TEM Orig-Sender: {rbirkhah-at-uceng.uc.EDU}:ddn:wpafb Orig-Author: {Ronald Henry Birkhahn {rbirkhah-at-uceng.uc.EDU} }:ddn:wpafb ----------------------------------------------------------- Hello, I was wondering if anyone had any experience with x-TEM of 6H-SiC,3C-SiC, on each other and Si. We are trying to look at the interface between SiC-Si and 6H-3C. Any thoughts on preparation (right now we cryo-ion mill) due to the hardness would be appreciated.
Thanks, Ron rbirkhah-at-uceng.uc.edu
---------------------- Replied Message Body ---------------------- To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: x-SiC TEM Orig-Sender: {rbirkhah-at-uceng.uc.EDU}:ddn:wpafb Orig-Author: {Ronald Henry Birkhahn {rbirkhah-at-uceng.uc.EDU} }:ddn:wpafb ----------------------------------------------------------- Hello, I was wondering if anyone had any experience with x-TEM of 6H-SiC,3C-SiC, on each other and Si. We are trying to look at the interface between SiC-Si and 6H-3C. Any thoughts on preparation (right now we cryo-ion mill) due to the hardness would be appreciated.
The UC stands for U. of Cincinnati, so I'm nowhere close to California. But I guess I don't know what is meant by "Tripod polisher". What is it and how does it work exactly? Is it anything like a dimpler?
Maybe someone can help me with my problem. As an electron-microscopist, I do have some troubles with embedding arteries and veins. I've tried to embed this arteries and veins of= =20 human umbilical cords, but didn't succeed completely.=20 The ultrathin sections show several holes and the tissue was damaged by shrinkage. We have tried to embedd the tissues in Unicryl,=20 but it was not satisfactory. Now we are planning to use Lowicryl HM20= .=20 Is there anyone out there who uses Lowicryl HM20 and who is experienc= ed in embedding arteries and veins (pieces). Do we have to use a special protocol? (fixation, cutting in pieces, embedding) Our goal is to look at the endothelial cell layer and the smooth muscle cells, together with the cell-contacts between these cells= =20 (gap-junctions). It will be an immuno-histological study, that's why we will try the Lowicryl HM20 plastic. What do you recomend, Lowicryl HM20 or Lowicryl KM4?
Thanks,=09=09Luc.
=C9=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD= =CD=CD=CD=CD=CD=CD=D1=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD= =CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=BB =BALuc Analbers =B3E-mail: Analbers-at-med.ruu.nl =BA =C7=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4= =C4=C4=C4=C4=C4=C4=C5=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4= =C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=C4=B6 =BAUniversity of Utrecht =B3 =BA =BAMedical Faculty =B3 Murphy's Law ??? =BA =BADept. of Medical Physiology=B3 =BA =BA & Sportsmedicine =B3 Well, Murphy was an optimist =BA =BAUniversiteitsweg 100 =B3 =BA =BA3584 CG Utrecht =B3 =BA =BAThe Netherlands =B3 LA =BA =C8=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD= =CD=CD=CD=CD=CD=CD=CF=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD= =CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=CD=BC
X400-Received: by mta bottom.ctd.anl.gov in /PRMD=ANL/ADMD= /C=US/; Relayed; Thu, 11 Aug 1994 02:47:52 -0500 X400-Received: by /PRMD=ESnet/ADMD= /C=US/; Relayed; Thu, 11 Aug 1994 02:47:51 -0500 X400-Received: by /PRMD=Switch/ADMD=Arcom/C=Ch/; Relayed; Thu, 11 Aug 1994 02:47:34 -0500 X400-Received: by /PRMD=SWITCH/ADMD=ARCOM/C=CH/; Relayed; Thu, 11 Aug 1994 02:48:31 -0500
John Mansfield North Campus Electron Microbeam Analysis Lab 413 SRB University of Michigan 2455 Hayward Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu or John.F.Mansfield-at-umich.edu Time: 11:10
Date:8/11/94 NC EMAL
Hi, my department (Materials Science and Engineering) would like to purchase a used SEM for the undergraduate teaching lab. It only needs to be a basic SEM, we'd like to get as new an instrument as possible. Budget is tight though and we'd like to pay less than $20K. Does anyone know of any instruments that fit our needs. Thanks.
In response to Steve Bill's concerns about back lighting the sample during Tripod Polishing:
We (South Bay Technology) have made several modifications to the the "L" bracket/Pyrex insert arrangement on a custom basis for customers. We are planning on introducing a variety of different "L" bracket and Tripod Polisher configurations in the next month. These new configurations will allow us to create "custom" packages that will more precisely fit specific applications. One of the first configurations will include an "L" bracket/Pyrex Insert arrangement that allows for easier back lighting of the sample.
I very much appreciate the comments and I encourage anyone else out there who has comments, questions or suggestions for new equipment or modifications to existing equipment to contact me. We are very interested in hearing from you and we will do our best to provide you with what you need. As for some of the more application specific information, I think I will leave that to our many Tripod Polishing experts out there on the Listserver!
Best regards-
David Henriks South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
Neuroscience List {neur-sci-at-net.bio.net} , Microscopy List {microscopy-at-aaem.amc.anl.gov} , Confocal Microscopy List {confocal-at-ubvm.bitnet} , Cell Bio List {cellbiol-at-net.bio.net} Message-Id: {Pine.3.05.1.9408111025.A23368-a100000-at-pauline.sdsc.edu} Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII
Please excuse this post if it is a little off-topic. What are the best Mac programs for compiling scientific references (all prices)? Sorry if this has been asked before. Thanks for all help.
Marc C. Brande, M.S. SD3D Email Discussion List: Live Brain Cell Biology in 3D All aspects of 3D Imaging San Diego 3D Imaging Group To subscribe/unsubscribe, send request to: 3840 Camino Lindo sd3d-request-at-sdsc.edu San Diego, CA 92122 To post a message to list,send message to: Email: BRANDE-at-SDSC.EDU sd3d-at-mailserver.sdsc.edu Voice: (619) 587-4830
If you have an Hitachi H7000/7110 TEM/STEM/SEM and want to use the bulk specimen holder for SEM work, you'll have found out that Hitachi charges $160.00 for each holder (I'm not kinding!) and they have to be special ordered from Japan since Hitachi USA doesn't keep any in stock.
As an alternate you can purchase JEOL 100cx SEM specimen holders for approx. $1.00/ea from Pelco, EMS, Fuller, etc. and modify them to fit the Hitachi bulk specimen rod in about 20 seconds with a pair of needlenose pliers and some wire cutters. The JEOL holders are about 6mm too long for the Hitachi rod and the end pieces are about 2 mm too tall, they are made out of strips of copper and are easily cut and bent.
Does anyone else out there use a Hitachi H7000/7110 for SEM Imaging?
I really love Endnote for storing and generating bibliographies. It is extremely easy to use (particularly if you use Microsoft Word), has powerful database search functions, allows you to put in notes about the reference, and can download reference information from sources such as medline (with the supplementary program, Endlink).
HELLO EVERYBODY WE,(ELECTRONMICROSCOPE UNIT, UNIVERSITY OF PRETORIA SOUTH AFRICA) ARE PARTICIPATING IN A CAMPAIGN TO PROMOTE NATURAL SCIENCES AMONGST CHILDREN (AGE 12-15 YRS) THE MAIN IDEA IS TO MAKE UP A "SCIENCE KIT" CONSISTING OF ITEMS RELEVANT TO THE VARIOUS NATURAL SCIENCE DISIPLINES EG. A SIMPLE CHEMISTRY KIT TO MAKE CRYSTALS. IS THERE ANYBODY OUT THERE THAT HAS BEEN THROUGH SUCH AN EXERCISE,OR THAT HAS SOME IDEAS? NEEDLESS TO SAY THAT THE BUDGET IS TIGHT! I WOULD APPRECIATE ANY CONTRIBUTIONS.
I FORGOT TO SAY THAT WE NEED TO CONTRIBUTE SOMETHING IN THE LINE OF MICROSCOPY TO THIS KIT;IF THAT DID NOT COME THROUGH CLEARLY IN MY 1ST MESSAGE! MY APOLOGIES !
For the last couple of years there has been a poster session devoted to that topic at the Society for Neuroscience annual meeting. I don't have the abstract booklets at my fingertips, but if you check in the programs you should be able to find the relevant abstracts and contact the authors directly. As I remember them from just a brief look, some of the kits and projects were very impressive, and covered a broad age range from elementary through high school.
Cheers,
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
I agree that the book NUMERICAL RECIPES has a very good explanation of the Wiener filter (and many other numerical techniques) -- but has anyone tried to actually use it ?
The occassional non-microscopy posting is not a big deal, however, keep it to a minimum. When I see things getting out of hand I'll speak up.. Remember there are a lot of other venues to get this type of information, but I realize that we all frequently face common problems relative to computers etc.. and it would therefore be nice to see what colleagues are using to solve simple problems (e.g. fonts, references, image formats etc...)
In message Fri, 12 Aug 1994 13:00:56 -0400, "Nestor J. Zaluzec-Argonne Nat. Lab." {ZALUZEC-at-anlemc.msd.anl.gov} writes:
} From: SMTP%"-at-ANLVM.CTD.ANL.GOV:Edbasga-at-USCN.BITNET" 11-AUG-1994 } 11:35:53.58 To: ZALUZEC } CC: } Subj: FEG BSE and EDS advantages? } } Message-ID: {9408111201296D3.ADSK-at-USCN.USCN.UGA.EDU} (UMass-Mailer 4.04) } Date: Thu, 11 Aug 94 12:02:02 EST } From: Edbasga%USCN.BITNET-at-ANLVM.CTD.ANL.GOV } Subject: FEG BSE and EDS advantages? } To: zaluzec-at-anlemc.msd.anl.gov } } We are considering replacing our SEM with a new one. } Our main work is in biological applications for a multi-user } environment at a Medical School. We will be dealing with cells } in culture and molecular biology applications. Dental materials } will also be examined. Since this is an acquisition that we will } have for 10-15 years I feel that a basic instrument with an FEG } would be a more sensible choice than a full blown W/LaB6 set up } (EDS, BSE, and cryostage). If we are locked into a W/LaB6, I feel we } will be unable to pursue low voltage, high resolution applications } especially in the cryo area. } } My major selling point is that the advantage of the higher beam } current density provided by an FEG at low voltages will outshine } any instrument with a conventional emittor. The additional detectors } and cryostage could always be added later to the FEG but a W/LaB6 } cannot be upgraded easily to an FEG. } } I have experience working with several FEG's in SEI mode both at } ambient and cryogenic temperatures. I know an FEG is the best } for high resolution (} 30,000x) for these applications. } } I don't have much experience with EDS or BSE imaging modes with FEG. } I think that the increased beam current density will be an } added advantage for applications such as localizing heavy metal } stained areas of chromosomes and colloidal gold labelled antigenic } sites using both BSE and EDS especially in a cryo environment. } } Will an FEG, in fact, provide better spatial resolution for BSE } and EDS applications for low voltage ( {10kV) and high resolution } imaging. At least one vendor claims there is no advantage } afforded by a higher beam current density for increasing BSE } spatial resolution. } } I need to convince the administration that FEG is worth the } extra cost in the long run. } } Thanks to all of you who respond. } } Ed Basgall, Ph.D. } Medical College of Georgia } Cellular Biology and Anatomy } Augusta, GA 30912-2000 } Ph: 706-721-3524 } FAX 706-721-6893 } ==================== Ed,
See the recent article by Heinzmann et al., 1994, SCANNING 16:, 241-245, and the literature cited there in for discussions on the use of BSE for biomedical specimens with a FESEM.
Thanks to all 18 people who responded to my question about the best mac programs for compiling scientific bibliographies (for journal articles). EndNote ($149) and EndNote Plus ($249) wins hands down (12votes).Also with sister product Endlink you can download refs from medline for integration. They will send you a video if you call for info! From: Niles and Assoc. 800 Jones St. Berkeley, CA 94710 Phone: 510-559-8592
Marc C. Brande, M.S. SD3D Email Discussion List: Live Brain Cell Biology in 3D All aspects of 3D Imaging San Diego 3D Imaging Group To subscribe/unsubscribe, send request to: 3840 Camino Lindo sd3d-request-at-sdsc.edu San Diego, CA 92122 To post a message to list,send message to: Email: BRANDE-at-SDSC.EDU sd3d-at-mailserver.sdsc.edu Voice: (619) 587-4830
Does anybody know of a need for the services of optical microscopy for intrepretation of mine tailings and other environmentally contaminated geological materials? In our lab we are working on a characterization protocol with this emphasis and need to know if it is a viable way to go before investing more time on the project. Thanks Clarissa
Hi, I'm looking for software that will let me design and control experiments on the Macintosh. Basically I'd like to have some kind of c like language that I could "write" the experiment in and have the program "run" this protocol. For example, loop over n trials where each trial consists of displaying 2 images and having the subject select via touch screen the correct image (where correct is defined in the protocol). I have no need to connect to the outside world via TTL's or A/D. I just need a flexible application that allows me to design and run different experimental protocols. Example programs that are on the PC that I'm aware of are Cortex from NIH, Tempo a commercial app from Reflective computing, Mel (not sure who does this one). I'd like to know of any mac programs that do this sort of thing.
thanks Gary Leydon leydon%bruce-at-biomed.med.yale.edu
Regarding the characterization protocol for mine tailings. If you have access to a SIMS, it would probably help you a great deal. Not only can it detect impurity levels down to the ppm or in some cases ppb levels, it can also discriminate isotopes, so if you mean by environmentally unfriendly - radioactive - it may help even further, although the operator probably wouldn`t be terribly excited.
I am personally partial to the Codonics NP-1600, largely because I have one. At $2.00 per shot, it produces prints that rival photographs in resolution, tonal range, and color saturation. It's the only printer I've yet seen that produces true blacks and true whites. The really nifty thing though is its convenience and accessibility. It attaches to an ethernet network and receives print requests via TCP/IP. Its set up as an FTP client, and you just FTP graphics files to it. I've yet to find a format it doesn't know. These features make it software and driver- independent, so its directly available to anyone with an account. Price is about 11K.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
Greetings, I have been involved with elemental analysis of ~10 um diameter calcium carbonate particles. There is interest in looking at the changes in surface charge by some analytical technique. Mass spec, nmr,atomic asorbtion, etc have been ruled out for various reasons. My question to the microscopy group is, would ATM or STM be of any use? If so, by what method could I get somebody to analyze these samples? What would the cost be and how rapidly could it be done? Thanks in advance.
************************************************* _______________________ * * | | * * | _____ ______ | * Charles J. Butterick (Chuck) * |__| | | |__| * Electron Microscopy Center * | | * Department of Cell Biology and Anatomy * ______| |______ * Texas Tech University Health Sciences Center * | __ __ | * Lubbock, Texas 79430 * |__| | | |__| * USA * | | * Phone 806 743-1633 voice * | | * 806 743-1219 fax * | | * Email emccjb-at-lubb.ttuhsc.edu * | | * * __| |__ * * |_______| *************************************************
Subject: Time:3:24 PM OFFICE MEMO German glass coverslips Date:8/16/94
We need to purchase 25mm round German glass coverslips (preferably 1.5mm thickness) for neuronal cell cultures. Can anyone steer us to a supplier? Mike
Mike_Schwartz-at-qm.yale.edu Fax 203-785-5263
Michael Schwartz, Ph.D. Section of Neurobiology Yale University School of Medicine 333 Cedar St. New Haven, CT 06510
} We need to purchase 25mm round German glass coverslips (preferably 1.5mm } thickness) for neuronal cell cultures. Can anyone steer us to a supplier? } Mike
You could try:
Hugh Cartwright 2407 Walter Zimny Drive Posen, IL 60469
(708) 489-1101
Suppliers of 1" round glass slides part# 458 $37.23/100 slides (plain with ground edge)
Ed Vicenzi tel (609) 258-1464 office Princeton University tel (609) 258-1406 lab Princeton Materials Inst. fax (609) 258-6878 70 Prospect Ave. Princeton, N.J. 08540-5211 email: vicenzi-at-phoenix.princeton.edu
Does anybody know of a good book dealing with morphometric calculations? Not having done math (especially calculus) for a number of years, I need to know of a formula for determining the area and volume of plasma membranes in thin sections. Also, if using the formula for the area of a sphere, does the formula indicate the surface area or the internal area? I remember the formula but which area it indicates I've forgotten. Thanks, Phil 8-{)
Our lab (Materials Science and Technology) is looking into the possibility of acquiring a scanning infrared microprobe. Presently, our only capability with respect to infrared analysis is a standard FTIR which is equipped with a diamond cell option for microanalyses. We have considered purchasing an FTIR-microscope attachment to increase our capabilities and to compliment our SEM/TEM and XRD facilities. Recently, we have seen information about a relatively new microanalysis system called a Scanning Infrared Microprobe. This technique apparently provides high resolution molecular microanalysis and morphological mapping of surfaces, bulk materials, etc.
Does anyone know anything about this technique/instrument that they could share with us? And if anyone has purchased one of these systems, are they satisfied or disastisfied, and why? All information is greatly appreciated. Thank you, Eliesh O'Neil Elizabeth (Eliesh) G. O'Neil Research Scientist I GTRI/EOEML Baker 271, 925 Dalney Street Atlanta, GA 30332-0827 (404) 853-0590 (office) (404) 894-6199 (FAX)
In message Wed, 17 Aug 1994 11:50:25 -0400, rutledge phil {prutle1-at-umbc.edu} writes:
} Does anybody know of a good book dealing with morphometric calculations? } Not having done math (especially calculus) for a number of years, I need } to know of a formula for determining the area and volume of plasma } membranes in thin sections. Also, if using the formula for the area of a } sphere, does the formula indicate the surface area or the internal area? } I remember the formula but which area it indicates I've forgotten. } Thanks, } Phil } 8-{) } ============== Martin W. Steer's book UNDERSTANDING CELLSTRUCTURE (1981, Cambridge University press) is a good and easy-to-understand book with many examples and illustrations. M.V. Parthasarathy Section of Plant Biology, 228 Plant Science Building Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734; Fax: 607-255-5407 E-Mail: mvp2-at-cornell.edu
In message Wed, 17 Aug 1994 11:50:25 -0400, rutledge phil {prutle1-at-umbc.edu} writes:
} Does anybody know of a good book dealing with morphometric calculations? } Not having done math (especially calculus) for a number of years, I need } to know of a formula for determining the area and volume of plasma } membranes in thin sections. Also, if using the formula for the area of a } sphere, does the formula indicate the surface area or the internal area? } I remember the formula but which area it indicates I've forgotten. } Thanks, } Phil } 8-{) } ============== Phil, We can either talk about area (a 2 dimensional quantity) or volume (a 3 dimensional quantity) the two are not interchangeable. The surface area of a sphere of radius r is 4*pi*r*r. (four pi r squared). The volume of a sphere of radius r is 4*pi*r*r*r/3 (four thirds pi r cubed). CBB
In message Wed, 17 Aug 1994 11:50:25 -0400, rutledge phil {prutle1-at-umbc.edu} writes:
} Does anybody know of a good book dealing with morphometric calculations? } Not having done math (especially calculus) for a number of years, I need } to know of a formula for determining the area and volume of plasma } membranes in thin sections. Also, if using the formula for the area of a } sphere, does the formula indicate the surface area or the internal area? } I remember the formula but which area it indicates I've forgotten. } Thanks, } Phil } 8-{) } ============== Phil, We can either talk about area (a 2 dimensional quantity) or volume (a 3 dimensional quantity) the two are not interchangeable. The surface area of a sphere of radius r is 4*pi*r*r. (four pi r squared). The volume of a sphere of radius r is 4*pi*r*r*r/3 (four thirds pi r cubed). CBB
My own uses are biological (Centre for Food and Animal Research, Agriculture and Agri-Food Candada) but I have used the SpectraTech instrument for infrared microspectroscopy. The Irus system software is under continuous upgrade, and the company (esp. John Reffner) extremely helpful in working out problems and new applications. They have an instrument installed at Brookhaven National Laboratory (using the National Synchrotron Light Source, which is much more powerful than conventional sources for IR) and are starting to do some very interesting work. I myself am still very much a neophyte at IR microscopy/spectroscopy, but for more info. contact Gwynn Williams at Brookhaven National Laboratory, Associated Universities Inc., Upton, New York, 11973. Have fun, and happy hunting. Shea Miller REs (last line is a typo... sorry!)
Phil- If you want to determine volume (a three dimensional parameter) from a thin section (essentially a two dimensional field) it seems to me you will need stereologic calculations to do the conversion from 2D measures to estimates of 3D parameters. The same would also appear to be true for determining area of plasma membrane. Although it is a 2D parameter, the membrane rarely stays in the plane of section and so its surface area must also be inferred from your thin section measurements. There are a number of good books and articles on the subject. My own favorite is Ewald Weibel's 1979 book Stereological Methods (Academic Press). Volume 1 is a practical treatment of the subject without too much math. Robert Bolender also has a book and computer program on the subject. He is a very readable author. journal articles by either of these two would also be helpful, in particular Weibel's 1970 Int Rev Cytology paper volume 26:235. Papers by Gundersen, Elias, Hyde, DeHoff, Cruz-Orive may also be helpful. Finally Elias and Hyde's 1983 A Guide to Practical Stereology (Karger Publishing- I think?) may be useful.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Pathology * * Bowman Gray School of Medicine of Wake Forest University * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Wed, 17 Aug 1994, rutledge phil wrote:
} Does anybody know of a good book dealing with morphometric calculations? } Not having done math (especially calculus) for a number of years, I need } to know of a formula for determining the area and volume of plasma } membranes in thin sections. Also, if using the formula for the area of a } sphere, does the formula indicate the surface area or the internal area? } I remember the formula but which area it indicates I've forgotten. } Thanks, } Phil } 8-{) }
Greetings, I'm interested in finding good, contemporary texts on TEM/SEM in French. Is anyone able to point me in the right direction? Many thanks!
Dwight Beebe IRBV, Dept. de sciences biologiques beebed-at-ere.umontreal.ca Universite de Montreal Voice:514-872-4563 4101, rue Sherbrooke est FAX:514-872-9406 Montreal, PQ H1X 2B2 Canada
There have been several good suggestions on this listserver regarding the use of LaB6 cathodes in the JEOL 2000 TEM. As a manufacturer of LaB6 cathodes, FEI has learned a lot over the years about how to best operate LaB6. Much has been covered, but I would like to make one further suggestion that may be helpful. We have found that it is beneficial to recheck your operating point often after start up. In the massive electron guns found in high voltage systems such as the JEOL 2000, it takes a while for the entire gun to reach thermal equilibrium when the cathode is first heated. Therefore, when you first find your operating point, the gun may not have reached thermal equilibrium. As the gun continues to heat up and finally reaches thermal equilibrium, less heat is transfered from the cathode to the gun, and your cathode will over heat. I recommend that the operating point be rechecked every 15 minutes for the first hour of operation. You will probably find that the filament setting needs to be reduced during that first 15 - 60 minutes of operation. This holds true for most high voltage TEMs (200kV and up) of any make, and also to smaller TEMs, to a lesser degree.
An additional option that may be helpful in the use of your TEM would be to try the FEI LaB6 Mini Vogel Mount (MVM) cathode on your next cathode change. We are currently showing good results in the 200 and 400 kV JEOL TEMs, as well as in many others. If anyone would like further details on our LaB6 cathodes, please contact me directly.
Best Regards
Locke Christman FEI Company 7451 N.E. Evergreen Parkway Hillsboro, OR 97124-5830 (503) 640-7518 lac-at-feico.com
Although probably not very appropriate for your applications, there is a v. useful book entitled "Initiation a la microscopie electronique par transmission" published by the Societe Francaise de Mineralogie et de Cristallographie, ed. C. Willaime. They are at Tour 16, 4 Place Jussieu, 75252 Paris CEDEX 05.
There has been a few questions & postings recently which tread on the gray area of this server and I've gotten several offline questions and comments on these. Let me share my thoughts with all of you on this.
First, let me remind everyone that ALL comments and question on microscopy and microanalysis are welcome on this server regardless of who posts them. I especially encourage commerical firms to read and if appropriate reply to posting which may need a special bit of information. This is a valuable part of what we are trying to foster. There have been many valuable posting from commmerical firms about solving problems and answers to questions and allow me to thank those that have participated.In the course of these replies it is appropriate to identify oneself and mention if a person or entity is a commerical firm or not especially if a particuliar product is mentioned. So far everyone has very carefully kept to this request and I am pleased.
There have been a few postings which border the gray area and I get occasional off-line questions about these and their nature.
Mostly they are replies for information on questions like: "Does anyone know if a company exists that does/sells XYZ for a fee" since, the request was posed by a subscriber I have no problems with a commerical firm saying "Sure we do/have that, and here's a brief synopsis, contact us off-line for more details" with the appropriate credit lines etc... I feel that this is within the bounds of answering a question and making the information on the existance of a company or service known to the subscribers. Most of the time everything that has been posted has been fine.
There have been the occasional posting which tread the line and I'd appreciate everyones cooperation here. When I see something that is inappropriate I do contact the author and let them know.
You should all also know that alot of the postings from commerical firms first send their reply to me for comment, especially when they mention a product or service, and I (and you should) appreciate their cooperation. Good examples are the recent discussions about FESEM's and LaB6.
So bear with the gray areas and I'll try to keep an eye on things, if you see something which appears strange just Email me and I'll investigate.
Just a reminder to no direct "advertising" of products or services should be conducted. Credit lines should be kept simple and direct to avoid any impression that your trying to sell something. It will make my life alot easier.....
Thanks in advance.... Nestor
P.S. I see no need for further discussion on this issue on the server. If you want to comment just contact me offline at:
Calcium localization in biological samples can be a problem unless the samples are properly prepared. Calcium phosphate or carbonate is very quickly lost under acid or even slightly alkaline fixes. If fixation must be used, try fixing at pH 7.5 - 8. Heavy metals such as osmium and uranium will also solubilize calcium, thus they should be avoided. Calcium also tends to be somewhat mobile during the fixation process and tends to translocate throughout the tissue, giving you localization artefact. For this reason, for calcium localization I routinely snap- freeze the tissue, then freeze-dry and embed in resin. Ultrastructural preservation is of course compromised, but if localization is what you want, this seems to be the best way.
Hope this helps.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
} As a materials scientist I am asking for help with a biological microscopy } problem. } } I recently had a user bring along some sections of biopsies in which he was } interested in finding calcium by EDX in the STEM.. The samples had been } prepared traditionally, namely: } } GTA 2.5%, PFA 2.0% Fix } OsO4 fix } dehydration in ethanol and propylene oxide } Uranyl Acetate en bloc stain } Epon embed } Section } } We found no calcium. I am reminded of a rather similar problem } several years ago when a user was studying embryonic mollusc shells, } and insisted calcium had to be present, but we could not find it. } } Does the traditional method of preparing samples remove calcium?
Yes. Suggest your user that he/she par their rubbish someplace other than your microscope. } Does anyone have a method of preparing samples that would retain } the calcium? } Yes again. Rapid freezing followed by cryoultramicrotomy. In some cases, for qualitative analysis, freeze substitution may be used, instead of cryoultramicrotomy.
} Any help would be much appreciated. } } ********************************************************* } * Anthony J. Garratt-Reed * } * Massachusetts Institute of Technology, Rm. 13-1027 * } * Cambridge, MA 02139, USA * } * * } * Ph: 617-253-4622 * } * Fax: 617-258-6478 * } ********************************************************* } }
Tony Garratt-Reed asked about Ca in biological samples. One problem could be wrong expectations of what is in the tissues. I have run into this problem *a lot*. As much as I explain that for concentra- tions of some millimolar XMA has no trouble with Ca, but for the physiological concentrations in the micromolar range XMA will not detect it, the message does not get through. Embryonic molusk may be mostly chitin or some such material which has no Ca in it. As far as I know, the normal prep methods will remove only the Ca++ solute from the cytoplasm and/or organelles; hydroxyapatite is not affected. Treatment with pyroantimonate will precipitate the Ca and it is visible in the EM, so you have a possible test for whether there is Ca in the sample after all. Also, examination of frozen, hydrated material or material lyophylized in situ in the EM (by heating to -110C for 24 hrs, -100C for 24 hrs and -90C for 24 hrs) which will reduce the mass thickness by about 80%, can el- iminate preparation artefacts for any Ca which is not volatile (essentially all of it). Good luck. Yours, Bill Tivol
In message {Chameleon.940819134408.tonygr-at-emlab.mit.edu} Anthony Garratt-Reed writes: } As a materials scientist I am asking for help with a biological microscopy } problem. } } I recently had a user bring along some sections of biopsies in which he was } interested in finding calcium by EDX in the STEM.. The samples had been } prepared traditionally, namely: } } GTA 2.5%, PFA 2.0% Fix } OsO4 fix } dehydration in ethanol and propylene oxide } Uranyl Acetate en bloc stain } Epon embed } Section } } We found no calcium. I am reminded of a rather similar problem } several years ago when a user was studying embryonic mollusc shells, } and insisted calcium had to be present, but we could not find it. } } Does the traditional method of preparing samples remove calcium? } Does anyone have a method of preparing samples that would retain } the calcium?
Anthony,
I would say that any calcium that was in the bio-systems you mention was washed out from the tissue samples during the above preparation, as most of it was not bound to the tissues in any way.
Alternatives: 1.freezing preparations: a. freeze-dry. b. freeze-substitution; followed by resin infiltration and sectioning.
2. Calcium precipitation in situ using the antimonate method. Tissues are treated with potassium antimonate solutions during fixation to precipitate calcium in situ, followed by the usual embedding techniques. There is always a question about transport of calcium away from its original location during any of these preparations, so keep an open mind.
The cool thing about the antimonate method is that, using EDS analysis, you can check for co-precipitation of Sb and Ca in your sections. Otherwise, you have to assume that any precipitates you obtain in your sections are the ones where the calcium is located; dangerous without the independent confirmation by EDS. Of course, the question about how accurate the localization is, is still there. But hopefully, you can at least precipitate it out within cells, at least, without having all of it washed out.
An uncool thing about this method is that the Sb L-series x-ray peaks lie right smack dab over the Ca K-series x-rays, but actually that just makes the analysis more fun because you get to use that peak deconvolution software that you've got; you've got to strip off the Sb L-series to reveal the Ca K-series beneath - make sure your energy axis is accurately calibrated when you collect these spectra.
Here are two references for the antimonate method. The first also includes EDS analysis of precipitates:
1. An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique, Robert D. Slocum and Stanely J. Roux, The Journal of Histochemistry and Cytochemistry, Vol 30 No. 7, pp. 617-629, 1982. Contains EDS analysis and tannic acid/antimonate procedure.
2. Localization of Ca++ containing antimonate precipitates during mitosis, Susan M. Wick and Peter K. Hepler, The Journal of Cell Biology, volume 86, August 1980, pp. 500-513.
Both of the above have more references to the technique.
3. I have a project in my lab right now doing an antimonate technique on plant cells and they seem to be getting resonable results. Soon I will send you their protocol.
Good luck!
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
I am looking for suggestions to prevent the clumping of cilia during processing for SEM. A buffer rinse prior to fixation didn't seem to help. I have thought of using a dilute Triton x-100 wash to try to remove the mucous layer prior to fixation, and was wondering if anyone has a favorite %. All suggestions welcome.
__________________________________________________________________ Randy Nessler rnessler-at-emiris.iaf.uiowa.edu The University of Iowa Central Electron Microscopy Research Facility
In message Fri, 19 Aug 1994 09:04:34 -0400, "Ronald M. Anderson" {ron-anderson-at-VNET.IBM.COM} writes:
} We have a Sorvall MT2-B microtome in need of a complete overhaul. } Is there someone near southeastern New York that anyone can recommend? } } Please direct responses to Phil Flaitz pflaitz-at-vnet.ibm.com offline. } Thank you. We will summarize and post as appropriate. } } 8-) } ========== For Sorvall MT 2B service in NY State try the following:
Bill McGee MICROTOME SERVICE Co. 7568 Florian Way Liverpool, NY 13088 Telephone: (315) 451-1404 M.V. Parthasarathy Section of Plant Biology, 228 Plant Science Building Cornell University, Ithaca, NY 14853. USA Tel: 607-255-1734; Fax: 607-255-5407 E-Mail: mvp2-at-cornell.edu
I am having a problem with fogging of histology slides after coverslip mounting. It appears to be the formation of tiny water droplets after mounting, like fine condensation. It appears to be above the focussing plane of the section. This is happening despite 3x3mins. dehydration in 100% ethanol (after 70% and 95% stages), hemo-de, histoclear or xylene clearing agents, and the fact that this is exactly the same protocol I have used for years, and used succesfully with the present lots of clearing agents and mounting medium (Permount) last Fall. Could summertime in upstate New York (increased humidity, although I'm working in a very well air-conditioned lab) cause this level of effect ? (This is a new environment for me.) Has anybody even run into this problem before, and did they find a solution ? Any suggestions would be greatly appreciated.
Patrick D. Reynolds Department of Biology Hamilton College Clinton, N.Y. 13323 Ph. (315) 859-4723 Fax: (315) 859-4807 e-mail: preynold-at-hamilton.edu
Patrick; I possed a similar question a couple of months ago. What kind of slides are you using??? Are they silane coated?? Do you coat your own or buy them pre-coated??? We have reduced the problem quite a bit by using isopropanol for dehydrating but still have the problem at times. It also appeared after years of working but have not found out why, except that we switched suppliers of slides (went from coating our own to usinf pre-coated slides). As can see from my address it is not just a problem of Upstate New York. I worked in Toronto for 7 years and never had the problem. Let me know what you find out and if you can solve it.
Yours Mark Elliott, PhD Pulmonary Research Lab, St. Paul's Hospital Vancouver BC Canada
} We have a Sorvall MT2-B microtome in need of a complete overhaul. } Is there someone near southeastern New York that anyone can recommend? } } Please direct responses to Phil Flaitz pflaitz-at-vnet.ibm.com offline. } Thank you. We will summarize and post as appropriate. } } 8-) } RMC, Inc. bought the old Sorvall, then DuPont, line and still services the MT-2Bs and other ultramicrotomes. I don't have my list of service people in your area here, but the main office in Tucson can be reached at (602) 889-7900. Old MT-2Bs never die, they just need an occasional tuneup!
Tina Weatherby Carvalho Biological EM Facility University of Hawaii
We are currently looking for a laser attachment for a light microscope for single cell ablation. I would like to hear from anyone who is currently doing this to discuss the various systems on the market, and to get your advise and opinion.
Ben August Dept. of Neurology, Tissue Culture Laboratory U of Wisconsin-Madison.
Without having had the experience myself (yet), I would put in a vote for the ethanol being the source of your clouding problem. For "non-critical" uses, we have purchased a cheap, bulk ethanol - 20 gal, presumably 100%. We have used it to rinse glass plates, and found that it leaves a cloudy residue behind - like water spots. Sound familiar?...
I think I'll buy the good stuff from now on. Good luck!
David Morilak Dept Pharmacology Univ Texas Health Science Center San Antonio
The cilia are coated wikth mucopolysaccharides and glycoproteins. You might want to try a protease or other cleaving enzyme. Triton will dissolve lipids, which aren't your problem, but it might help keep any digestion residues on the cilia from adhering to each other.
Let me know what works.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
On Fri, 19 Aug 1994, randy nessler wrote:
} } I am looking for suggestions to prevent the clumping of cilia } during processing for SEM. A buffer rinse prior to fixation didn't seem } to help. I have thought of using a dilute Triton x-100 wash to try to } remove the mucous layer prior to fixation, and was wondering if anyone has } a favorite %. All suggestions welcome. } } __________________________________________________________________ } Randy Nessler } rnessler-at-emiris.iaf.uiowa.edu } The University of Iowa Central Electron Microscopy Research Facility } } }
I've always used 10- min. changes in the 100% ethanol (3 baths) and 3 baths in xylene, 10 min. each. Change solutions every 2 weeks or 200 slides. Its usually not as humid here in the Pacific NW, but this has always worked. This works for sections up to 40 micron vibratome sections as well as 80 micron celloidin sections.
Incidentially, try DPX from Gallard and Schlesinger, as a coverslipping medium. It won't prevent fogging from residual water, but its nice stuff to work with.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Anthony Garrett-Reed, Given how labile Ca is, I would be surprised if it wasn't lost in preparation. Only the insoluble precipitates like bone can be counted on to hang around during processing. You most likely will have to try freezing and frozen-sectioning of hydrated tissues. Maybe freeze-fracture & examination in an SEM with EDX. There was an excellent paper in the early '80's about doing this with insect tissue for soluble ions (Na, K, etc.), but naturally I forget the ref, and my copy is buried who knows where. Maybe someone else remembers it... Phil Oshel poshel-at-luc.edu
In reply to Anthony Garratt-Reed I strongly agree with Professor Somlyo's response to your query and add a small note of caution about a possible pitfall in the use of pyroantimonate protocols. If you consider the diffusion coefficients of calcium and antimony you may come to the conclusion that the precipitate may tell you more about the point of entry of antimony into the cell or tissue than it does about the normal site of calcium. Further, calcium is not the only biologically important element precipitated by antimony. Dispersive x-ray of cryopreps is the way to go. Best luck! David
David B. Slautterback Anatomy Department 264 Bardeen Labs UW-Madison Voice 608-262-1609 Email dbslautt-at-macc.wisc.edu
Does anyone know whether there is a mailing list/usenet group dedicated to collagen/extracellular matrix. We are looking for a specialist in this field.
Nikon makes digital control backs for their cameras. I think one is available for the F3 (you'll also need a film winder/motor drive). I know they made one for the 8008.
The following query has been forwarded to the group for its consideration
From gsumnich-at-sunstroke.sdsu.edu Tue Aug 23 08:39:41 1994
I am considering purchasing a Cohu 1300 series color video camera to do some video microscopy. I would like to be able to use the integration capabilities of this camera but the one unit I have found to do this (model 440 A Frame Store, Colorado Video Inc.) seems a bit pricey. The unit must control integration time of the camera, capture the video output from the camera, store the image, and send it to the computer at 30 frames/sec. Does anyone know any other packages that will do this?
I have had good success in dissolving mucous on mucosal surfaces using 1% dithiothreitol (DTT) prior to fixation. Its most effective on mucous with many disulfide linkages, as do most vertebrate mucouses.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
The Temple City address and phone number I have for South Bay Technology appear to no longer be valid. Does anyone have a current address and phone for them? Are they still in business?
TIA
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
} The Temple City address and phone number I have for South Bay Technology } appear to no longer be valid. Does anyone have a current address and } phone for them? Are they still in business? } } TIA } } Glen MacDonald } Hearing Development Laboratories RL-30 } University of Washington } Seattle, WA 98195 } (206)543-8360 } glenmac-at-u.washington.edu } } } They moved to San Clemente a few years back. South Bay Technology 1120 Via Callejon San Clemente, CA 92672 714 492-2600 phone 714 492-1499 fax
Edward Goo Associate Professor Department of Materials Science and Engineering University of Southern California Los Angeles, CA 90089-0241 ekgoo-at-mizar.usc.edu (213) 740-4426 phone (213) 740-7797 fax
Thanks for the address, and to all who responded directly to me. This will help someone out looking for a diamond saw. I had an address off a diamond saw that was several years old.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu
Message-Id: {MAILQUEUE-101.940823135607.448-at-vanlab.paprican.ca} To: *Anatomo Pathologie {anapat-at-reks.uia.ac.be}
Hello, If you do not find a list server to assist you, there is a company in the US. which specializes in finding experts in any scientific area in order to access information from them. They charge a fee and I'm not sure how they would handle a single search situation, but the company name is Teltech and they may be reached as follows:
Teltech 2850 Metro Drive Minneapolis, MN 55425-1566
or
SUPPORT-at-US.TELTECH.COM
Good luck, Laurie
______________________________________________________________________ Laurie Frederick, A.SC.T. PAPRICAN Corrosion Control Group 3800 Wesbrook Mall The Pulp and Paper Research Vancouver, B.C. Institute of Canada Canada V6S 2L9
I am rebuilding a Denton DV502 evaporator for use in our undergrad EM course. If anyone out there has an out-of service DV502, or any of the following parts available, please contact me directly. Please *do not* reply to the list. -Expanded metal guard for 12"x12" bell jar -Manual lift for 12" x 12" bell jar -Glow-discharge feed-through and ring TIA Julian Smith III Dept of Biology Winthrop University Rock Hill, SC 803-323-2111 (vox) 803-323-2246 (fax) smithj-at-winthrop.edu
} Date sent: Tue, 23 Aug 1994 11:44:05 -0500 (CDT) } From: COOK-at-AAEM.AMC.ANL.GOV } To: MICROSCOPY-at-AAEM.AMC.ANL.GOV } Subject: 35 mm time lapse control
} Nikon makes digital control backs for their cameras. I think one is availabl } e } for the F3 (you'll also need a film winder/motor drive). I know they made on } e } for the 8008. }
The multi-function control back for the 8008 and 8008S is NIKON MF-21. Interval-timmer function Long time exposure Auto bracketing Freeze focus Recently purchased for $165.00. Adorama (800)223-2500.
*************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
Embedding adipose tissue Does anyone have any suggestions on how to embed adipose tissue and to retain all the lipid? I want to do some straight foward morphological studies. I have tried a few different methods, and no matter what I do, in at least half of my blocks, the center part has lost the lipid. Also, how easy is it to freeze and do ultracryosections on this tissue? Thank-you in advance. Jeanne
I work with monolayers of differentiated intestinal cells which I grow on glass coverslips. We routinely fix and embed in plastic resins like Epon, JB-4, or LR Gold but I am having trouble getting a good paraffin embedding technique. I tried embedding scrappings of the monolayers in agar prior to paraffin infiltration but was not overwhelmed by the results. any ideas gratefully appreciated.
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
The suggestion here in regards to the problem of "Water Dropletts" appearing under the coverslips in recent years, is the use of Re-cycled Xylene in the final xylene rinse and coverslipping step. Re-cycled xylene is fine for the earlier staining & processing steps but just isn't clean enough for the final step, use fresh xyxlene. These are the words of my friendly HT, HTL (Histotech.) Federal guidelines necessitated the use of re-cycled xyxlenes for many lab in the last couple of years (which explains why everything was fine in years gone by.)
It's worked for us, let us know if it works for you.
Richard E. Edelmann Electron Microscopy Facility Supervisor Marshall University - School of Medicine Huntington, West Virginia
My humble replies to several of your queries: 1. I don't think you will ever retain all of your lipids. We have measured phospholipid, cholesterol, and triglycerides before and after fixation (not an easy thing to be sure you are doing right, and probably some artefacts induced) or freezing. In both cases there is selective loss of particular lipid species. Lipids will leach out of intracellular stores in prolonged exposure to glycerol or sucrose as a cryoprotectant as well. This is probably of concern only if you are doing specific anlysis of lipids. For straightforward morphological studies do you really need to retain all lipids, or merely retain the volume and location in an identifiable way (i.e. know where the lipids were?). What is the exact problem with simple fixation with prolonged osmium postfix? Has the central portion of your lipid vacuole collapsed? If not, unless you want to try and measure what species of lipid is present, why worry? It is an artefact most of us crazy enough to study lipids have learned to accept.
2. Ultrarapid freezing followed by freeze substitution is probably the easiest method to preserve the bulk of the lipids in a reasonably similar location and volume. I haven't worked with adipose tissue, but atherosclerotic lesions have a high fat content and this has worked, even for quantitative ultrastructural analysis. the key is good freezing. There are a number of books on the subject and freezing has been discussed in this forum. In my experience, you will probably need to slam freeze if your tissue is of any size at all. Others with more experience than me can probably comment better.
3.Ultracyromicrotomy is a difficult thing (IMHO) and not for the faint of heart or inexperienced. If you go this route, hook up with an expert. Here again, good initial freezing is critical. The comment about getting an expert to help (physically) is probably also applicable to 2 above.
On 24 Aug 1994, Jeanne Barker wrote:
} Embedding adipose tissue } Does anyone have any suggestions on how to embed adipose tissue and to retain } all the lipid? I want to do some straight foward morphological studies. I } have tried a few different methods, and no matter what I do, in at least half } of my blocks, the center part has lost the lipid. } Also, how easy is it to freeze and do ultracryosections on this tissue? } Thank-you in advance. } Jeanne } } }
I suggest phosphate buffered fixatives, and a rapid dehydration schedule with cold acetone. Reference: Mary Williams et al in a pulmonary journal.
On 24 Aug 1994, Jeanne Barker wrote:
} Embedding adipose tissue } Does anyone have any suggestions on how to embed adipose tissue and to retain } all the lipid? I want to do some straight foward morphological studies. I } have tried a few different methods, and no matter what I do, in at least half } of my blocks, the center part has lost the lipid. } Also, how easy is it to freeze and do ultracryosections on this tissue? } Thank-you in advance. } Jeanne } } }
After reading the recent discussion of the LaB6 problem I was still unsure of the exact nature of the cathode failure. Am I correct in assuming that the reason the cathode was taken out of service was that the emission current became too high? Try looking at the base and the sub-base of the cathode. If there is evaporated material between the mount structures at either of these surfaces, you may have an electrical path which will show up as an increased emission current.
I started last year a program of salvaging old computers of no use in the states anymore, by helping nuns of the order of Our Lady of Perpetual Help. These nuns picked me up from the barrios of the Dominican Republic and put me on the right path to chose what I am today.
Old XT and slower machines (primarily MS-DOS based) of no use to us because of newer software demands are still powerful teaching tools for undeveloped countries (e.g., one of the school teaching typing does not have a single electric typewriter; they run 10 or more schools). I brought down with me last year to the Dominican Republic two XT we had here at Tulane in my department, and I am planing to return this fall with other machines. So far I have two electric typewriters and two potential XT 4.7 MHz.
I know firsthand that goods intended for the poor do not reach them first if donations are sent through certain agencies (e.g., my poor parents had to purchase milk and cheese from the soldiers handling donations from the US when I was 12). Thus, I am personally supervising transfer of goods and making sure that those goods stay in the hands of the nuns, and are exclusively used for teaching the kids of the 20 schools they operate throughout the Dominican Republic.
If you know of sources that could donate machines needing very minor repairs additions of floppies, etc. I would be most grateful. I am paying the cost personally for the expenses of repair and shipment, and there is no monetary gain whatsoever on my part from this; just a moral debt payment!
Those with machines to donate can contact me via internet or:
Hi everybody, I'm working with the ultrastructure of the biofilm which have colonised the tubing from dental chair unit. The biofilm consist of bacteria and water organisms which have adhered to the material surface. So if you can see the "biofilm.sea.hqx" and if you know the water organism, please send me his name. Thank you very much,
I would like to know if someone has experience sectioning cerebellum culture specimens. My problem is that this specimen seems to degrades new diamond knives by introducing striations on a new edge. Does anybody know wether cerebellum is inherently rough on the knives or if it may be my embeding techniques at fault. The embeding medium I use is LR white. Any comments would be appreciated.
-I would like to know if someone has experience sectioning cerebellum culture -specimens. My problem is that this specimen seems to degrades new diamond knives -by introducing striations on a new edge. Does anybody know wether cerebellum is -inherently rough on the knives or if it may be my embeding techniques at fault. -The embeding medium I use is LR white. Any comments would be appreciated.
I have cut lots of brain and some of it was cerebellum I have never had any problems. I cut mostly Poly Bed, some of it hard, some of it very soft, never any problem. Are your blocks to soft? You could be getting debris on the knife edge. I would try cleaning it first.
I have had problem with cutting cultured cells. That problem was that someone who changed the culture was using cheap glass pipets that had not been fire polished. Small pieces of glass got in the culture. I only had to hit one to know I had a problem. I alway processed the dishes with plastic pipets, but someone else grew the cells.
I would look for possible sources of contamination at each step. Check to make sure that new plastic items are used whenever possible. Are the grown on glass cover slip? Could something have gotten in the stocks of plastic componates? Look for any source of glass or dust or any like that. Someone else in your lab might be less careful that they should be.
} I would like to know if someone has experience sectioning cerebellum culture } specimens. My problem is that this specimen seems to degrades new diamond } knives } by introducing striations on a new edge. Does anybody know wether cerebellum } is } inherently rough on the knives or if it may be my embeding techniques at fault. } The embeding medium I use is LR white. Any comments would be appreciated. } } B. E. Mesa } 177000.1040-at-COMPUSERVE.COM
I have no experience with LR White, but I know that normal cerebellum, processed with routine methods, will not cause problems. I'd check the plastic to see whether it is too hard or inconsistent. Are you having problems with only a few blocks, or several batches of embedded specimens?
One thing you might try is to section with glass and see whether you get knife marks, and, if you do, maybe see whether they come from the plastic or the tissue. Another thing you might try is to section a blank block of the plastic, even turn the block around and section the opposite end. That way, you'd have blank plastic as close as possible in characteristics as what the tissue is in.
Good luck,
John chandler-at-lamar.ColoState.EDU Fort Collins CO
Hello Paul If you are looking for an basic introduction to dislocations, a book by Hull and Bacon is a good text, titled Introduction to Dislocations, Permagon Press, 1984. If you wish to know about images of dislocations in TEMs and how to characterise them, the monographs by Edington are useful (Practical electron microscopy in materials science, JW Edington, Van Nostrand Reinhold Company) as is Loretto's Electron beam analysis of materials, Chapman and Hall, 1994. Good luck Ciara
In message {199408251930.PAA12250-at-neuro.duke.edu} Larry Hawkey writes:
} B. E. Mesa 177000.1040-at-COMPUSERVE.COM wrote} } } -I would like to know if someone has experience sectioning cerebellum culture } -specimens. My problem is that this specimen seems to degrades new diamond } knives
Larry Hawkey responded:
} I have had problem with cutting cultured cells. That } problem was that someone who changed the culture was } using cheap glass pipets that had not been fire polished. } Small pieces of glass got in the culture. I only had to } hit one to know I had a problem. I alway processed the } dishes with plastic pipets, but someone else grew the cells. } } I would look for possible sources of contamination at each } step.
I only want to underscore what Larry Hawkey said about using glass pipets, as I have seen the same problem with tiny glass fragments carried into the resin and running into them with a knife: Bink! followed by: -at-#%*##&-at-#!!!!!
If you must use glass pipets, make a special set of cleaned pipets to use exclusively for your handling of fixatives, dehydrations series liquids, and embedding resins. I rinse them, inside and out, with distiled water, then ethanol, let air dry.
--
Gib Ahlstrand Electron Optical Facility University of Minnesota Dept. Plant Pathology 495 Borlaug Hall St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX giba-at-puccini.crl.umn.edu
Message-Id: {9408252103.AA26010-at-riker.ml.wpafb.af.mil} To: WALCKSD:ml:wpafb, SCHELTFJ:ml:wpafb Subj: Re: Dislocation reference Orig-Author: {Ciara Mullan {mullanc-at-mcmail.cis.mcmaster.ca} }:ddn:wpafb ----------------------------------------------------------- Hello Paul If you are looking for an basic introduction to dislocations, a book by Hull and Bacon is a good text, titled Introduction to Dislocations, Permagon Press, 1984. If you wish to know about images of dislocations in TEMs and how to characterise them, the monographs by Edington are useful (Practical electron microscopy in materials science, JW Edington, Van Nostrand Reinhold Company) as is Loretto's Electron beam analysis of materials, Chapman and Hall, 1994. Good luck Ciara
As part of a surplus purchase of SEM specimen boxes, I have a few dozen used aluminum stubs that appear to go with Coates & Welter. Having an ISI/Topcon 'scope, I have no use for them. If you'd like them mailed to you, please contact me directly. Please *do not* reply to the list. Julian Smith III Dept of Biology Winthrop University Rock Hill, SC 803-323-2111 (vox) 803-323-2246 (fax) smithj-at-winthrop.edu
To all, re: recycled xylenes-- This problem can also be avoided by the use of Histo-Clear (from National Diagnostics), and a similar compound from Fisher & who else? It's made from essential oils of citrus plants & is non-toxic (& smells like oranges). I've used it a bunch, & it works as well as xylene for histological procedures. [I have no financial interests in Nst. Diag.] Phil Oshel poshel-at-luc.edu
Just a reminder of a discussion that we had several months ago.
DO NOT post images to this listserver. If you want to ask for help about a particuliar image then you should upload the image to an FTP site where individuals (who want) can download the file and try to give you a hand.
I do not want random individuals filling up everones Email boxes with huge unrequested files!
I'm sure there will always be someone who will look at the file but remember in all likelyhood a good fraction of the people on this server may not be interested in your data.
I have sent a copy of this notice to the individual who recently posted an image to the server. I see no need for further discussion.
John Mansfield and I have been considering establishing an upload area on an anonymous FTP site for people who do not have the ability to do this on their own. We will keep you updated as things progress. At the moment the anonymous FTP site both he and I maintain are READ ONLY..
I suggest you get in touch with my colleague Dr. Subash Chandra (e-mail: sc40-at-cornell.edu Phone: 607-255-4137) regarding protocols for preparing plant suspension cells for analysis with SIMS. Dr. Chandra is an expert in the application of SIMS technology to analyse biological material.
Please do not send multiple images over the net, especially on the same day. This will jam the email account if the quota is limited or full fill the floppy disc if you are using Eudora.
Ya Chen ---------------------------------------------------------------------------- ------ | Assistant Researcher/Cryo-SEM Coordinator | Integrated Microscopy Resource (IMR)-- | An NIH Biomedical Research Center | University of Wisconsin-Madison | 1675 Observatory Drive #167
Appearently Phillips no longer supplies the neccesary parts to put a 35mm camera on a Phillips 300. Anyone who has one that is not being used and would like to sell can either e-mail me at cal-at-ssnet.com with the info and I will forward, or contact Dr. Moudrianakis at 410-516-7305. Thanks, Cal
} Embedding adipose tissue } Does anyone have any suggestions on how to embed adipose tissue and to retain } all the lipid? I want to do some straight foward morphological studies. I } have tried a few different methods, and no matter what I do, in at least half } of my blocks, the center part has lost the lipid. } Also, how easy is it to freeze and do ultracryosections on this tissue? } Thank-you in advance. }
I don't know if you want to do optical microscopy or electron microscopy, but if you want optical microscopy, you may fix with 4% paraformaldehyde in 1% calcium chloride and 1% cadmium chloride. The material must be postfixed with 0,5 osmium tetroxide in sacarosis and embedded in glicol methacrilate. Cut the sections with glass knives. Good luck! ============================================================================= Francisco Javier Hernandez Blazquez | Universidade de Sao Paulo - Faculdade de | e-mail fjhblazq-at-fox.cce.usp.br Zootecnia e Engenharia de Alimentos | Voice: 55 195 616122 r. 265 Departamento de Ciencias Basicas/Histologia| r. 268 Av. Duque de Caxias Norte, 225 CP 23 | Fax: 55 195 611689 CEP 13630-000 Pirassununga (Sao Paulo) | BRAZIL | ==============================================================================
I am looking for a fluorescence illuminator (or a vertical illuminator that I could modify into fluorescence) for my Olympus Vanox microscope or for the labs' BH-2. If you have a used Olympus illuminator that you would be willing to part with, please contact me directly. Please *do not* reply to the list. TIA Julian Smith III Dept of Biology Winthrop University Rock Hill, SC 803-323-2111 (vox) 803-323-2246 (fax) smithj-at-winthrop.edu
As one of the people who has been having the water droplets problem-this is a NEW problem which has just arisen. We have been using Histo-Clear for years with no problem-worked great, as did Hemo-De, the Fisher product. It has only been in the last couple of months that this has arisen, and it happens no matter which clearing agent we use-Histo-clear, Hemo-de or xylene. We used fresh xylene from the manufacturer- we have no access to a still to recycle our xylene. We also changed from using ethanol for dehydrating to using isopropanol, this has helped a bit but we still occasionally get the water droplets. We have increased our dehydrating and clearing times to 5 min in each alcohol and 3x10 min changes in clearing agent which reduces the problem greatly but increases the time it takes for coverslipping. We still do not know why this has started to occur after using standard methods for years.
Mark Elliott, PhD UBC-Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver BC Canada
I am trying to apply an historic stain named "Movats Stain", one of the components in this witches brew is "Wood Stain Scarlet". However no one has any idea who to get it from. The original suppliers got rid of their last batch in 1971! does anyone out there have a musty bottle in their archive, or perhaps know of a supplier or an equivalent dye
Thanks Simon C. Watkins Director SBIC University of Pittsburgh Pittsburgh PA 412-648-3051
Hi, does anyone know of any replacements for OsO4 as a conductive stain for biological specimens. Coating of complex extracted plant tissues for HRSEM with thin (2-3nm) layers of Pt or Cr has not proved enough to avoid charging. This is the case at both low and high kV. As a result I'm having to use OsO4 but would prefer not to, due to its deleterious effect on actin and in masking antigenicity. Ive tried to use uranium but with little success. Any comments or hints? Cheers
Peter
Peter Vesk, E.M. Unit,F09 University of Sydney NSW 2006 phone: 61 2 692 2351 (overseas) fax: 61 2 552 1967 peter-at-emu.su.oz.au
Peter, You might consider binding the OsO4 with thiocarbohydrazide (TCH) to increase the conductivity. See Platt-Aloia, K.A. and Thomson, W.W. 1980. Aspects of the three-dimensional intracellular organization of mesocarp cells as revealed by scanning EM. Protoplasma 104: 157-165. (and references within). Hope this helps.
Mike Postek and I experimented with this procedure about 15 years ago, using variations of the O-T-O (osmium thiocarbohydrazide osmium) technique. Using thiocarbohydrazide as a ligand, ruthenium red worked at least as well, and as I recall, gold chloride also worked. You might try other ligands as well, such as carbohydrazide and hydrazine instead of TCH...its more stable than the other 2, but not as effective. Hope this helps.
-=W.L. Steffens=- College of Veterinary Medicine University of Georgia
In message Tue, 30 Aug 1994 03:21:13 -0400, peter-at-emu.su.oz.au (Peter Vesk) writes:
} Hi, does anyone know of any replacements for OsO4 as a conductive stain } for biological specimens. Coating of complex extracted plant tissues for } HRSEM with thin (2-3nm) layers of Pt or Cr has not proved enough to avoid } charging. This is the case at both low and high kV. As a result I'm having } to use OsO4 but would prefer not to, due to its deleterious effect on } actin and in masking antigenicity. Ive tried to use uranium but with } little success. Any comments or hints? } Cheers } } Peter } } Peter Vesk, } E.M. Unit,F09 } University of Sydney } NSW 2006 } phone: 61 2 692 2351 (overseas) } fax: 61 2 552 1967 } peter-at-emu.su.oz.au } ============================== Peter,
Try 0.5-1% tannic acid followed by unranium salt solution.
Peter- have you tried using a light tannic acid treatment at the prefix stage to protect actin etc against osmium? Helps both preservation and conductive staining, and may cut down the number of TA/Os cycles later....at low kV I suspect it should be enough by itself if you mount the specimen really carefully. Method in Stowe Fukudome and Tanaka, Cell Tiss Res 1986. Wont help the antigenicity though, I suppose!
Sally ---------------------------------------------------------------------- Sally Stowe | Australian National Univ. Facility Coordinator Canberra, AUSTRALIA ANU Electron Microscopy Unit | Ph 61 6 249 2743 Email stowe-at-rsbs-central.anu.edu.au | FAX 61 6 249 4891 -------------------------------------|-------------------------------- -
I have two questions for which I would appreciate some info back if possible. Also, I think they are applicable to list server goals. 1. Has anyone had any interaction with O 1. Has anyone had any interaction with OSHA or your own safety personnel concerning the interpretation of the prior apporval section of OSHA rule 29 CFR 1910.1450 (the so-called lab standard)? Has this caused anyone any problems?
In our institution, the literal interpretation of prior approval means that it is necessary to have the approval of the department head and the safety office for "any laboratory work with any hazardous substances." Prior approval shall "be required before implementing any new laboratory activities." Some people use the interpretation that prior approval is meant only for "exotic" chemicals. I would be interested in how other EM labs interpret the prior approval rule.
2. Does anyone know of an inexpensive on-line service which provides updated MSDS information? I am aware of a CD ROM service which will provide a CD ROM service on a quarterly basis but I would prefer on-line service.
John C. Wheatley Arizona State University CSSS PSB-234 Tempe, AZ 85287-1704 TEL: 602-965-3831 FAX: 602-965-9004
One of our students is attempting to determine the extent of live cells from the sapwood of tree cores. It has been suggested that 1% aqueous triphenyl tetrazolium chloride may be used to stain for both macroscopic and microscopic determinations of living cells. Is anyone aware of additional staining/microscopy procedures which may be used to make a reasonably accurate quantitative determination of the extent of live cells in sapwood? Richard Crang Professor Plant Biology UIUC (217) 244-3143
One of our students is attempting to determine the extent of live cells from the sapwood of tree cores. It has been suggested that 1% aqueous triphenyl tetrazolium chloride may be used to stain for both macroscopic and microscopic determinations of living cells. Is anyone aware of additional staining/microscopy procedures which may be used to make a reasonably accurate quantitative determination of the extent of live cells in sapwood? Richard Crang Professor Plant Biology UIUC (217) 244-3143
} I am looking for a low cost PC based SLOW SCAN frame grabber } for taking images from an SEM. Any ideas? } Thanks. } } Rob Thomson } EM Facility } Victoria University } Wellington } New Zealand
We are using the ImageSlave System developed by Meeco/Dindema in Sydney Aust. It allows image collection at the photograhic scan rate on our Hitachi S4100 FeSEM. It is also possible to use the memory photograph function via the ImageSlave. The cost is around $3000 (very approximate) AUD. It runs under DOS on a PC.
We have found the system to be very effective.
The address is:
MEECO HOLDINGS 10 Seville St. North Parramatta NSW 2151 AUSTRALIA
Tel + 61 2 630 7755 Fax + 61 2 630 7365
I hope this of use to you.
Colin
##################################################################### ******************************* * Logic is invincible because * 0------* in order to combat logic it * } ---|--- { * is necessary to use logic. * | * P.Boutroux * / \ ******************************* _/ \_
Colin Veitch Tel + 61 (0)52 27 5611 CSIRO Division of Wool Technology Tel + 61 (0)52 27 5891 (dir.) P.O. Box 21 Fax + 61 (0)52 27 5657 BELMONT Vic 3216 Australia
John C. Wheatley wrote} } 1. Has anyone had any interaction with OSHA or your own safety personnel } concerning the interpretation of the prior approval section of OSHA rule 29 } CFR 1910.1450 (the so-called lab standard)? Has this caused anyone any } problems?
We just had a meeting with the Safety Office here at Duke. They are meeting with all Medical Center personnel. This was a two hour session that covered fire safety, Radiation safety, and other things. Nothing was said about use of hazardous chemicals. As far as I know I can use any chemical I want to in my lab. (we are a research dept. not hospital or clinical.) I think that if I want to use radiation, I must clear it with the Radiation Safety office. This is for disposal more than use I think.
Our Safety office does little with training or clearing the use of Hazardous. The only thing they said was that lab personnel have the right to know what they are working with and their supervisor is responsible for telling them. Since I am the only one in the EM lab I guess it is my problem.
I would also like to see some on line service for MSDS information. I would think that some goverment office would provide that for free?
Hello! I am doing some EM on oysters and looking at protists in the tissue. I was wondering if anyone might have a good receipe for a fixative that would be good for fixing the oyster tissue and the protists. I currently use 2.5% glut in 0.2M cacodylate buffer, pH 7.4. Should the fix and the buffer rinses be made up in filtered sea water? What might be the best pH and osmolarity of the fix and buffer? TIA, Phil
} Our Safety office does little with training or clearing } the use of Hazardous. The only thing they said was that } lab personnel have the right to know what they are working } with and their supervisor is responsible for telling them. } Since I am the only one in the EM lab I guess it is my } problem.
If I'm not mistaken, you are also responsible for informing anyone who comes into the lab, especially if they will be doing any work, about hazards they might run into. Some of these issues come down to legal liability, in case of accident or injury.
It's not an easy topic, and I think we all would do well to be aware of the hazards around us, chemical, radiation, electrical, vacuum, etc., and not become too casual.
John chandler-at-lamar.ColoState.EDU Fort Collins CO
I definitely agree that the quality of calibration replicas has decreased over the last 10 years. We calibrate our microscopes every semester and go through many replicas as we have 75 students using them each semester and so many are distorted, of various contrasts, difficult to see, etc. It is almost if there are replicas being made of replicas. I have tried to check them in a calibrated light microscope and they definitely are of different sizes. I would appreciate knowing what the quality assurance is when making them and the statistical deviation of what the measurement should be claimed by the manufacturers.It sure makes for interesting curves!! Perhaps if the manufacturers who make them are listening, they could give us some input. Thanks, Judy M.
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
Microscopy Society of America contact is: Caroline Schooley Box 117 Caspar, CA 95420 707/964-9460 The program is in conjunction with the Lawrence Hall of Science in Berkeley and is in conjunction with the middle schools. judy m
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
I FORGOT TO SAY THAT WE NEED TO CONTRIBUTE SOMETHING IN THE LINE OF MICROSCOPY TO THIS KIT;IF THAT DID NOT COME THROUGH CLEARLY IN MY 1ST MESSAGE! MY APOLOGIES !
Message-Id: {1994Aug26.174159.898881934-at-ms.sjdccd.cc.ca.us} To: microscopy-at-anlemc.msd.anl.gov (microscopy)
To: microscopy
I definitely agree that the quality of calibration replicas has decreased over the last 10 years. We calibrate our microscopes every semester and go through many replicas as we have 75 students using them each semester. Many are distorted, of various contrasts, difficult to see, etc. It is almost if there are replicas being made of replicas. I have tried to check them in a calibrated light microscope and they definitely are of different sizes. I would appreciate knowing what the quality assurance is when making them and the statistical deviation of what the measurement should be, claimed by the manufacturers.It sure makes for interesting curves!! Perhaps if the manufacturers who make them are listening, they could give us some input. Thanks, Judy M.
Judy Murphy, PhD Dept. of Microscopy San Joaquin Delta College 5151 Pacific Ave Stockton, CA 95207
There have been some very good points made in this thread, especially in Bob Craig's response. Most research labs are far too relaxed on safety and training. Someone in every lab or department, especially where there are multiple users, particularly students, grad students, and clinical students, must be the designated safety officer. Specific issues like fire, chemicals, radiation, biological hazards, etc. can be delegated to individuals iwth expertisew, preferably who are actively using these agents. These people should have some authority along with this responsiblility. If someone is not competent with these agents, they shouldn't be allowed to use them. The University of Washington, at the command of the Seattle Fire Dept., requires that each "Chemical Use Area" i.e. laboratory, have a designated contact person who maintains an inventory of all chemicals stored in that area. The primary purpose is so that the fire dept. knows what to expect in case of emergency. The side effect has been that many labs, including our own, finally realized what they had on their shelves. In our case, many users had been planning experiments and purchasing chemicals without looking to see what was already available. the result was many duplications, outdated chemicals and chemicals that were improperly stored. I weeded the shelves, consolidated chemical purchases and created a Hypercard based inventory system. If anyone is interested in beta-testing the update to this inventory system, reply to me directly.
Further, and it is a pain, but the best first line of defense are procedure manuals. Write a page or two describing proper use, (realistic) hazards, and proper disposal for particularly hazardous chemicals. Include this in your notebooks with the recipes and procedures that use those chemicals. It takes time but each of us is directly in a position of risk from our own actions, as well as everybody else's. I've grouped many of these together for groups of compounds that are related or used for similar purposes. For example, our fixatives share a couple of pages of instruction. Osmium and DAB have their own guidelines.
With 20 or so users in our labs, we found it essential to create a "Main Brain" list. This one page lists various labs, equipment, procedures and reagents and gives the name of someone responsible for training in their use. Even faculty don't touch things until getting instruction from the Main Brain person.
Make it clear to all newcomers that they are expected to read these instructions and contact the Main Brain for whatever they do. Mistakes will still occur, but with less frequency and fewer consequences.
Glen MacDonald Hearing Development Laboratories RL-30 University of Washington Seattle, WA 98195 (206)543-8360 glenmac-at-u.washington.edu